ACETYLATION OF P45/NF E2 BY PCAF AND ITS CONSEQUENCES

造血是多能祖细胞获得谱系特异性表型并最终产生成熟循环血细胞的过程。 控制造血的环境信号最终由一组转录因子来解释，这些转录因子调节与谱系特异性功能相关的基因的表达。 该提案的长期目标是定义导致造血转录因子的调节和激活的信号事件。 该建议旨在表征乙酰转移酶PCAF对红细胞/巨核细胞转录因子NF-E2的调节。 发现NF-E2的造血亚基p45在高度保守的赖氨酸残基处被PCAF乙酰化。 这一观察形成了我们的假设，即p45的活性可能是由乙酰化调制的基础。 具体目标是：1）确定p45乙酰化的机制后果。 p45 a在参与DNA结合的CNC结构域中被PCAF乙酰化。 将确定p45乙酰化对DNA结合的影响。乙酰化还可以改变p45的构象，从而调节其与其他蛋白质的相互作用。 最后，缺乏乙酰化位点的突变型p45的转录活性将通过p45缺失的红系细胞中的基因互补来检查。 2)在巨核细胞和红细胞分化过程中监测p45的乙酰化。 为了建立分化细胞中p45乙酰化/脱乙酰化与p45靶基因活化之间的相关性，将产生特异性识别乙酰化p45的抗体。 包含体内乙酰化位点的肽将用于产生对乙酰化p45特异的抗体。将在白血病细胞系和经历分化的原代细胞中跟踪p45的乙酰化状态。 将检查诱导分化的化学物质和细胞因子对p45乙酰化的影响。总之，这些研究可能会产生重要的见解调节造血转录因子的乙酰转移酶。 由于这些酶提供了很好的药物干预靶点，因此这些知识可能有助于设计用于影响血液病患者基因表达和细胞分化的药物。

Hematopoiesis is the process in which pluripotential progenitors acquire lineage-specific phenotype and eventually give rise to mature circulating blood cells. Environmental signals that control hematopoiesis are ultimately interpreted by sets of transcription factors which regulate the expression of genes associated with lineage-specific functions. The long-term objectives of this proposal are to define the signaling events leading to the modulation and activation of hematopoietic transcription factors. This proposal aims at characterizing the regulation of the erythroid/megakaryocytic transcription factor NF-E2 by the acetyltransferase PCAF. The hematopoietic subunit of NF-E2, p45, was found to be acetylated by PCAF at a highly conserved lysine residue. This observation forms the basis of our hypothesis that the activity of p45 may be modulated by acetylation. The specific aims are to: 1) Determine the mechanistic consequences of p45 acetylation. p45 a is acetylated by PCAF in the CNC domain which participates in DNA binding. The effects of p45 acetylation on DNA binding will be determined. Acetylation could also change the conformation of p45 thus could modulate its interaction with other proteins. Finally, the transcriptional activity of mutant p45 lacking the acetylation site will be examined by gene complementation in p45-null erythroid cells. 2) Monitor acetylation of p45 during megakaryocytic and erythroid differentiation. To establish the correlation between p45 acetylation/deacetylation and the activation of p45 target genes in differentiating cells, antibodies specifically recognizing acetylated p45 will be generated. A pepetide encompassing the in vivo acetylation site will be used to generate antibodies specific for acetylated p45. The acetylation status of p45 will be followed in both leukemic cell lines and primary cells undergoing differentiation. The effects of both differentiation-inducing chemicals and cytokines on p45 acetylation will be examined. Together, these studies may yield important insights into the regulation of hematopoietic transcription factors by acetyltransferases. Since these enzymes present excellent targets for pharmacological intervention, this knowledge could be useful for the design of drugs used to influence gene expression and cellular differentiation in patients with hematological disorders.